Method of purifying Gro β or Gro α protein using antibodies

ABSTRACT

Two novel cytokines which are involved in an inflammatory response (inflammatory cytokines) are disclosed. The inflammatory cytokines have been isolated from activated peripheral blood, preferably monocytes adhered to plastic; or peripheral blood leukocytes induced with calcium ionophore and mezerin. The two cytokines are Gro β and γ, and their cDNA and amino acid sequences are disclosed. The expression of the genes in various cells is presented. Assay and diagnostic utilities using the cytokines; recombinant materials and procedures; purification procedures; and monoclonal antibodies to the cytokines are also shown.

RELATED APPLICATIONS

This application is a divisional application of U.S. appln. Ser. No.08/190,249, filed Jan. 31, 1994, a continuation application of U.S.appln. Ser. No. 07/590,223, filed Sep. 28, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of molecularbiology/immunology and diagnostic uses therein.

BACKGROUND OF THE INVENTION

The present invention involves novel cytokines, in particular Gro genesand proteins, which were derived from the cDNAs of induced peripheralblood cells, for example monocytes. Therefore, the following discussionpresents the background on cytokines, monocytes and the Gro genes andproteins.

A. Cytokines:

Cytokines are small molecular weight proteins that have a broad range ofcell regulatory activity both in vitro and in vivo. (For backgroundinformation, see Balkwill, F. R., et al., 1989, Immun. Today, 10:299).Some cytokines possess growth factor activity. Examples of cytokineswhich can inhibit cell growth or directly kill cells are interferons(IFNs), tumor necrosis factor (TNF), lymphotoxin (LT), the interleukin1s (IL1s) and transforming growth factor β (TGF-β). Most cytokines acton B or T cells at some stage in their response. An example of thephysiological effect of cytokines can be seen in the case of cytokineswhich regulate the inflammatory response by varying the T and Blymphocyte activation, chemotaxis, eicosanoid secretion and collagenproduction. Differential expression of these inflammatory mediators mayinfluence the perpetuation of chronic inflammation and fibroticmanifestations.

B. Monocytes:

Monocytes are directed to sites of infection by chemotactic factorsreleased as a result of inflammation or infection (Sporn, S. A., et al.,1990, J. of Immun., 144:4434-4441). The migration of monocytes into thetissue involves interaction with vascular endothelium and the subsequentmigration of the monocytes through the underlying basement membrane,during which the monocytes come into close contact with extracellularmatrix components and connective tissue cells (Harlan, J. M, 1985,Blood, 65:513; Wilkinson, P. C., et al., 1979, Curr. Top Pathol.,68:47).

Thorens, B., et al., 1987, Cell, 48:671 reported that adherence might beimportant in transcriptional expression of macrophage mediators ofinflammation and that adherence to different matrices could result inpreferential gene induction. Further, there is evidence forparticipation of cell adhesion molecules in developmental interactionbetween epithelial and mesenchymal cells influencing celldifferentiation (Sanders, E. J., 1988, Biochem. Cell Biol., 66:530). Ithas also been shown that within 30 minutes of monocyte adherence toplastic, a complex set of regulatory events is initiated as defined byrapid changes of mRNA levels of several proto-oncogenes and inflammatorymediators (Haskill, S., et al., 1988, J. of Immunol., 140:1690). IL-1Bβ,TNF-α and c-fos are rapidly elevated, whereas CSF-1 steady state mRNAlevels increase by 90 minutes. In contrast, expression of c-fms andlysozyme is rapidly down-regulated. These genes are modulated byadherence to different biologically relevant substrates (Eierman, D. F.,1989, J. of Immunol., 142:1970-1976).

Although high steady state mRNA levels of important mediators ofinflammation are rapidly induced by adherence, adherence by itself isinsufficient to cause efficient translation and secretion of IL-1β,TNF-α, or CSF-1 (Haskill, S., et al., supra). Activation by a secondsignal, such as bacterial endotoxin, is required for the secretion ofall three gene products. Thus, it is clear that signals derived from theact of adherence are likely to play a significant role in the activationand differentiation of monocytes allowing them to respond to infectionand to influence the local tissue environment (Sporn, S. A, supra).Recent studies indicate that adherence leads to a general activation ofnumerous genes involved in the early defense response and whoseexpression may be regulated by selective tissue/extracellular matrixinteractions. Id.

C. Gro Genes/Proteins:

Adherence of monocytes results in the rapid induction of high levels ofmostly transient mRNAs for various novel inflammatory mediator genes(Sporn, S. A., et al., supra). One of these novel clones, Gro α, had apredicted amino acid sequence similar to that reported for the originalGro gene product. Mapping studies of Gro (Anisowicz, A., et al, 1988,PNAS (USA), 85:9645-9649 and Richmond, A., et al.,1988, EMBO J,7:2025-2033) identified a unique Gro site at chromosome 4q21. The Grogene belongs to a gene super-family which encodes a set of relatedcytokines that includes NAP-1/IL-8 (Matsushima, K., et al., 1988, J.Exp. Med., 167:1883-1893; Schmid, J., et al., 1987, J. of Immunol.,139:250-256; Peveru, P., et al., 1988, J. Exp. Med., 167:1547-1559), andplatelet basic protein (PBP). PBP is the precursor of connective tissueactivating protein III (CTAP III), β-thromboglobulin (Castor, C. W., etal., 1983, PNAS (USA), 80:765-769), platelet factor 4 (PF4) (Deuel, T.F, et al., 1977, PNAS (USA), 74:2256-2258), γ-interferon-induciblepeptide (γIP-10) (Luster, A. D., et al., 1985, Nature (London),315:672-676), and macrophage inflammatory protein 2 (MIP-2) (Wolpe, S.D., et al., 1989, PNAS (USA), 86:612-616).

Gro was initially identified by its constitutive over-expression inspontaneously transformed Chinese hamster fibroblasts (Anisowicz, A., etal., 1987, PNAS (USA), 84:7188-7192). A related gene was identified inv-src transformed chicken cells (Sugano, S., et al., 1987, Cell,4:321-328; Bedard, P. A., et al., 1987, PNAS (USA), 84:6715-6719). Inexpression studies with normal fibroblasts, Gro showed early responsekinetics similar to c-fos, leading to the name Gro (growth regulated)(Anisowicz, A., et al., 1987, supra). Later, a protein with melanomastimulating activity (MGSA) (Richmond, A., et al., supra) was shown tobe encoded by Gro, and sequence similarity was reported with the murineearly response gene KC (Oquendo, P., et al., 1989, J. Biol. Chem.,264:4133-4137).

Preliminary studies showed that the Gro α gene was expressed in activeulcerative colitis disease, but not in the inactive tissue. (Isaacs, K.,et al., "Profiles of cytokine activation in inflammatory bowel diseasetissue: measurement of cDNA amplification", American GastroenterologicalAssoc. & American Assoc. for the Study of Liver Diseases, May 13-16,1990, Texas (Abstract)). On the other hand, disparity in expression ofthe Gro α gene was less in the case of active versus inactive tissuesfrom Crohn's disease. The expression of the Gro α gene in activeintestinal inflammation suggests a role of these cytokines in thepathogenesis of inflammatory bowel disease.

A cDNA designated MAD-2 was shown at a 1988 RES Conference. (Sporn, S.et al., "Isolation of Adherence Specific cDNA Clones from a MonocytecDNA Library" Society for Leukocyte Biology (RES), Washington, D.C.,Oct. 27-30, 1988; and in Sporn, S. A. et al., J. of Immunol, 1990,144:4434.) MAD-2 was isolated by differential hybridization from a cDNAlibrary which was prepared in the bacterial expression vector λgt10 byusing total RNA fom human blood monocytes adhered to plastic for 30minutes at 37° C. Sporn, S. A. et al., J. of Immunol, 1990, supra. MAD-2and human Gro were believed to be separate gene products based onSouthern blots analysis and differences in the amino acid sequences inthe carboxyl-terminus amino acids. Id. at 4440. At the Cytokine Workshopof Dec. 13, 1989, Hilton Head, Georgia, Ruth Sager presented the aminoacid sequences of both Gro β and γ.

Recently, two cDNAs for the human homologs of murine MIP-2 had beencloned from a library prepared from phorbol myristate acetate(PMA)--treated and lipopolysaccharide (LPS)--stimulated U937 cellsTekamp-Olson, P. et al., 1990, J. Exp. Med., 172:911. These cDNAs,designated MIP-2γ and MIP-2β are closely homologous to the cDNAsdisclosed in the pending patent application.

SUMMARY OF THE INVENTION

One aspect of the invention presents two novel inflammatory cytokinegenes, Gro β and γ, that have been found in peripheral blood cell cDNAlibraries.

Another aspect of the invention presents the cDNA and amino acidsequences of Gro β and γ and methods for obtaining them.

Another aspect of the invention presents a description of the expressionof Gro β and γ genes in various cells.

Another aspect of the invention presents the uses of Gro β and γ fordetecting agents which cause inflammation (hereinafter referred to asinflammatory agents), or agents which prevent or reduce inflammation(hereinafter referred to as anti-inflammatory agents).

Another aspect of the invention presents the use of Gro γ or β fordiagnosing cancer, preferably human colonic cancer in the case of Gro γ.

Another aspect of the invention presents recombinant Gro β and γ andprocedures for obtaining them.

Another aspect of the invention presents monoclonal antibodies againstGro β and γ.

Another aspect of the invention presents methods for purifying Gro β andγ proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS 1(A) and 1(B) show the cDNA sequences and predicted translationsequences of the open reading frames for Gro B and Gro y compared withGro α, noting positions of amino acid differences. Probes used forSouthern DNA transfer and primers used for polymerase chain reaction(PCR) analysis of the RNAs are underlined. Bold typed regions indicatethe positions of the ATTTA motif, and conserved regions between the 3human Gro isolates are boxed (beginning at bp848 for Gro α) as well as aregion common between human and hamster (bp930 Gro α).

FIG. 2 shows the Southern transfer analysis which demonstrated thepresence of three distinct Gro genes. Lanes 1-3, hybridization with theGro α, β and γ cDNA clones. Lanes 4-7, oligonucleotde hybridization todehydrated agarose gels (Caput, D., et al., 1986, PNAS (USA),83:1670-1674). Lane 4, oligonucleotide ML80; Lane 5, GM349; Lane 6, Groα specific oligonucleotide (GM350); and Lane 7, Gro β specificoligonucleotide GM272.

FIG. 3 shows the PCR analysis of Gro expression in different cells fromthe same individual or similar cells from different individualsstimulated by different signals. Lane 1, neutrophils; 2, lymphocytes;and 3, monocytes, each adhered for 45 minutes to fibronectin coatedplastic; 4, adherent monocytes stimulated with bacterial polysaccharide(LPS) for 4 hours; 5, monocytes stimulated under non-adherent conditionswith phorbol 12-myristate 13-acetate (PMA) for 4 hours; 6, endothelialcells stimulated with LPS; and 7, fresh biopsy of colonic carcinoma. Alldata are shown at 30 amplification cycles.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All of thesepublications and applications, cited previously or below are herebyincorporated by reference.

Although any similar or equivalent methods and materials may be employedin the practice or testing of the present invention, the preferredmethods and materials are now described.

A. Definitions

The term "Gro β" and "Gro γ" as used in relation to protein materialthroughout the present application and claims refer to protein materialshaving the amino acid sequences presented in FIG. 1(B) and theirfunctional equivalents. Two amino acid sequences are functionallyequivalent if they have substantially the same biological activities.Further included in the scope of "Gro B" and "Gro y" are theirpropolypeptide and mature polypeptide forms. FIG. 1(B) shows thepropolypeptides of Gro β and Gro γ which include their signal peptidesor leader sequence. Cells secrete mature polypeptides which have theirsignal peptides removed. For therapeutic or diagnostic uses, the maturepolypeptides are preferred over the propolypeptide. Further, differentsignal peptides can substitute for the naturally occurring ones. Themature polypeptide begins at about position 35 (Ala₃₅) for Gro β, and atabout position 34 (Ala₃₄) for Gro γ.

Independent of their biological activities, two polypeptides are alsoconsidered functionally equivalent if they have similar sequences ordisplay substantially equivalent sequences to the prolypeptide sequencesof Gro β or Gro γ in FIG. 1(B) or their mature polypeptide sequences.Preferably, two polypeptides are functionally equivalent when at leastabout 88% of their amino acids match over a defined length of the maturepolypeptides. For this calculation, there is a penalty of one wrongamino acid per mismatched or unmatched (deletion) residue.

Modified proteins are also within the contemplation of this patentapplication. These modifications may be deliberate, for example, such asmodifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in the hosts.

Further, as is the case for all proteins, the precise chemical structuredepends on a number of factors. As ionizable amino and carboxyl groupsare present in the molecule, a particular protein may be obtained as anacidic or basic salt, or in neutral form. All such preparations whichretain their activity when placed in suitable environmental conditionsare included in the definition. Additionally, the primary amino acidsequence may be augmented by derivatization using sugar moieties(glycosylation) or by other supplementary molecules such as lipids,phosphate, acetyl groups and the like, more commonly by conjugation withsaccharides. The primary amino acid structure may also aggregate to formcomplexes, most fiequently dimers. Certain aspects of such augmentationare accomplished through post-translational processing systems of theproducing host; other such modifications may be introduced in vitro. Inany event, such modifications are included in the definition so long asthe activity of the protein is not destroyed. It is expected that suchmodifications may quantitatively or qualitatively affect the activity,either by enhancing or diminishing the activity of the protein invarious assays.

Individual amino acid residues in the chain may also be modified byoxidation, reduction, or other derivatization, and the protein may becleaved to obtain fragments which retain activity, Such alterationswhich do not destroy activity do not remove the protein sequence fromthe definition.

Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the sequence duringtranslation can be made without destroying the activity of the protein.Such substitutions or other alterations result in proteins having anamino acid sequence which falls within the definition of Gro β and γproteins.

Among other things, the Gro proteins are involved in inflammatoryresponse. The terms "inflammatory cytokine" is intended to includewithin its scope the Gro β and γ proteins specifically recited herein,as well as all their functional equivalents.

Similarly, the terms "Gro β" and "Gro γ" as used in relation to DNAsequences throughout the present application and claims refer to the DNAsequences presented in FIGS. 1(A) and (B) and their functional homologs.Also within the scope of this patent application are DNA sequences orfragments which encode protein or peptide having substantially the samebiological activities as Gro β or γ. These DNA sequences or fragmentsare hereinafter called functional homologs.

Further, within the definition of functional homologs and independent oftheir biological activities, are DNA sequences which are substantiallyhomologous to the DNA sequences of Gro β or γ presented in FIGS. 1(A)and 1(B) and the DNA sequences of their functional homologs. Two DNAsequences are "substantially homologous" when at least about 94% of thenucleotides match over the defined length of the DNA sequences,preferably the DNA sequences are without the leader sequence. Sequencesthat are substantially homologous can be identified by comparing thesequences using standard software available in sequence data banks, orin a Southern hybridization experiment under, for example, stringentconditions as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See. e.g.,Maniatas, et al., 1982, DNA Cloning: A Practical Approach, Vols. I & II,(Glover, D. N., ed. 1985); and Hanes, B. D., et al., 1985, Nucleic AcidHybridization.

In Example 1 below, Gro β and γ were derived from the cDNA of activatedor induced (the term "activation" and "induction" are usedinterchangeably) peripheral blood cells. The preferred cells areperipheral blood leukocytes and most preferably monocytes. The preferredactivation method in the case of peripheral blood leukocytes are bymeans of mezerein and calcium ionophore. The preferred activation methodin the case of monocytes is by means of adherence to extracellularmatrixes or other substrates. The specific example shown was byadherence to plastic. However, other cell lines or other sources for thedevelopment of either the material from which the Gro β and Gro γ arethereafter isolated, or the Gro β and Gro γ themselves, are contemplatedherein and the present invention is accordingly not limited For example,within the contemplation of this patent application are cells which areactivated or induced, for example by mitogens such as LPS or PMA oradherence to other extracellular matrixes or by viral infections, andthereby express Gro β or Gro γ. (An example of viral induction is seenin Dudding, L., et al., 1989, J. Immunology, 143:3343, "HumanCytomegalovirus Infection Stimulates Expression of Monocyte-AssociatedMediator Genes".) Tumor cells which express Gro β or Gro γ are alsowithin the contemplation of this patent application.

Alternate means, such as genetic replication which may be conductedaccording to principles of recombinant technology that are well known inthe art are contemplated herein in accordance with the presentinvention.

Thus, Gro β and Gro γ nucleic acid sequences may be obtained byscreening genomic or cDNA library from any cell. The DNA may also beobtained by synthesizing the DNA using commonly available techniques andDNA synthesizing apparatus. Synthesis may be advantageous because uniquerestriction sites may be introduced at the time of preparing the DNA,thereby facilitating the use of the genes in vectors containingrestriction sites not otherwise present in the native source. Further,any desired site modification in the DNA may be introduced by synthesis,without requiring further modification of the DNA by mutagenesis.

An "antibody" is defined herein to include any immunoglobulin, includingantibodies and antigen binding fragments thereof (e.g., Fab, F(ab)₂,Fv), which bind a specific epitope. The term encompasses, inter alia,polyclonal, monoclonal, single chain and chimeric antibodies, the lastmentioned are described in further detail in U.S. Pat. Nos. 4,816,397and 4,816,567.

B. Establishment of a cDNA Library

A full length cDNA sequence that encodes the inflammatory cytokine maybe obtained using molecular biology techniques well known in the art,with the noted exceptions detailed below.

Several procedures are available for identifying the cytokine cDNAsequences. The preferred procedure is to generate a library using RNAisolated from peripheral blood cells, more preferably peripheral bloodleukocytes and monocytes, but a library can be generated from virtuallyany source of biological material that expresses the inflammatorycytokine; indeed, cDNA libraries can even be purchased commercially.Monocytes and peripheral blood cells are the preferred startingmaterials because the adherence of monocytes to an appropriate surface,and the activation of the peripheral blood leukocytes by calciumionophore and mezerein, induce the expression of the inflammatorycytokines.

An illustrative procedure for making a cDNA library containing theinflammatory cytokine sequences consists of isolating total cytoplasmicRNA from a suitable starting material, and further isolating messengerRNA therefrom. The latter can be further fractionated into Poly (A+)messenger RNA, which in turn may be fractionated further still into Poly(A+) messenger RNA fractions containing inflammatory cytokine messengerRNA. The messenger RNA can then be reverse transcribed and cloned into asuitable vector to form the cDNA library.

More specifically, the starting material (i.e., tissue, cells) is washedwith phosphate buffered saline, and a non-ionic detergent, such asethylene oxide is added in an amount to lyse the cellular, but notnuclear membranes, generally about 0.3%. Nuclei can then be removed bycentrifugation at 1,000×g for 10 minutes. The post-nuclear supernatantis added to an equal volume of TE (10 mM Tris, 1 mMethylenediaminetetraacetic acid (EDTA), pH 7.5) saturatedphenol/chloroform (1:1) containing 0.5% sodium dodecyl sulfate (SDS) and10 mM EDTA. The supernatant is re-extracted 4 times and phase separatedby centrifugation at 2,000×g for 120 minutes. The RNA is precipitated byadjusting the samples to 0.25 M NaCl, adding 2 volumes of 100% ethanoland storing at -20° C. The RNA is then pelleted at 5,000×g for 30minutes, washed with 70% and 100% ethanol, and dried. This representsthe total cytoplasmic RNA.

Attentively, total cytoplasmic RNA may be isolated using the guanidineisothiocyanate-cesium chloride method as described by Chirgwin ea.,1979, Biochem., 18: 5294.

Polyadenylated (Poly A+) messenger RNA (mRNA) can be obtained from thetotal cytoplasmic RNA by chromatography on oligo (dT) cellulose (J. Avivet al., 1972, PNAS, 69:1408-1412). The RNA is dissolved in ETS (10 mMTris, 1 mM EDTA, 0.5% SDS, pH 7.5) at a concentration of 2 mg/ml. Thissolution is heated to 65° C. for 5 minutes, then quickly chilled to 4°C. After bringing the RNA solution to room temperature, it is adjustedto 0.4 M NaCl and slowly passed through an oligo (dT) cellulose columnpreviously equilibrated with binding buffer (500 MM NaCl, 10 mM Tris, 1mM EDTA, pH 7.5) The flow-through is passed over the column twice more,and the column washed with 10 volumes of binding buffer. Poly (A+) mRNAis eluted with aliquots of ETS, extracted once with TE-saturated phenolchloroform and precipitated by the addition of NaCl to 0.2 M and 2volumes of 100% ethanol. The RNA is reprecipitated twice, washed once in70% and then 100% ethanol prior to drying. The poly (A+) mRNA can thenbe used to construct a cDNA library.

cDNA can be made from the enriched mRNA fraction using oligo (dT)priming of the poly A tails and avian myeloblastosis virus (AMV) reversetranscriptase employing the method of H. Okayama et al., 1983, Mol. CellBiol. 3:280.

Other methods of preparing cDNA libraries are, of course, well known inthe art. One, now classical, method uses oligo (dT) primer, reversetranscriptase, tailing of the double stranded cDNA with poly (dG) andannealing into a suitable vector, such as pBR322 or a derivativethereof, which has been cleaved at the desired restriction site andtailed with poly (dC). A detailed description of this alternate methodis found, for example, in U.S. Ser. No. 564,224, filed Dec. 20, 1983,and assigned to the same assignee.

Using the partial amino acid sequence of Gro α (see FIGS. 1(A) and (B)),and known codon redundancies thereto, several DNA oligonucleotide probesmay be synthesized and used to screen the cDNA library.

A preferred method by which a cDNA clone that encodes the inflammatorycytokines may be identified is to employ a cDNA library that is producedusing RNA obtained from induced monocytes or peripheral blood cells, andto detect individual clones that differentially hybridize to cDNA probesproduced using RNA from induced and uninduced monocytes or peripheralblood cells. Clones that preferentially hybridize to cDNA probesproduced from induced but not uninduced monocyte or peripheral bloodcell RNA will contain cDNAs that encode the inflammatory cytokines ofthe instant invention.

cDNA inserts may be sequenced using known techniques. The preferredtechnique is to subclone the inserts into an appropriate vector, anexemplary vector being pGEM blue (Promega Biotec. Madison, WisconsinCorp.), and sequence the double stranded DNA using the dideoxy chaintermination method described by Sanger et. al., 1977, PNAS (USA),74:5463. Sequencing is conveniently performed using commerciallyavailable kits, preferably the SEQUENASE™ sequencing kit produced byUnited States Biochemical Co. Cleveland, Ohio, and using suitableprimers, such as T7 and SP6 obtainable from Promega Biotec. Madison,Wis., and sequence specific primers.

C. Immunological Procedures for Detecting the Inflammatory Cytokines

The presence of the inflammatory cytokine can be ascertained by a numberof immunological procedures known in the art. Examples of usefulprocedures include: the "competitive" procedure described in U.S. Pat.Nos. 3,654,090 and 3,850,752; the "sandwich" procedure described in U.S.Pat. Nos. RE 31,006 and 4,016,043; and the "double antibody" or"capture" procedure. In the capture procedure, three antibodies areused: Ab₁, Ab₂, and Ab₃. The procedure may be represented by thefollowing equation: Ab₁ +Cyt+Ab₂ +*Ab₃ =Ab₁ Cyt Ab₂ *Ab₃. Cyt representsthe inflamatory cytokine, and *Ab₃ represents labelled Ab₃. Ab₁ and Ab₂recognize the inflammatory cytokine, whereas the labelled *Ab₃recognizes Ab₂. Ab₁ is bound to a support. The presence of theinflammatory cytokine is detected by the resulting labelled complex ofAb₁ Cyt Ab₂ *Ab₃.

In each of the procedures, the inflammatory cytokine forms complexeswith one or more antibody(ies) or binding partners and one member of thecomplex is labelled with a detectable label. The fact that a complex hasformed and, if desired, the amount thereof, can be determined by knownmethods applicable to the detection of labels.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine and auramine. A particular detecting material is anti-rabbitantibody prepared in goats and conjugated with fluorescein through anisothiocyanate.

The inflammatory cytokine or its binding partner(s) can also be labelledwith a radioactive element or with an enzyme. The radioactive label canbe detected by any of the currently available counting procedures. Thepreferred isotope may be selected from ¹⁴ C, ¹³¹ I, ³ H, ¹²⁵ I and ³³ S.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colonmetric spectrophotometric,fluorospectrophotometric or gasometric techniques. The enzyme isconjugated to the selected particle by reaction with bridging moleculessuch as carbodiimides, diisocyanates, glutaraldehyde and the like. Manyenzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labelling material and methods.

The above procedures and their applications are all familiar to thoseskilled in the art and accordingly may be utilized within the scope ofthe present invention.

D. Recombinant Procedures

The following describe general recombinant procedures, which could bemodified by those skilled the art for obtaining recombinant Gro β and γ.

1. Suitable Hosts, Control Systems and Methods:

In general terms, the production of a recombinant form of Gro β or γtypically involves the following:

First, a DNA encoding the mature protein (used here to include allmuteins); the preprotein; or a fusion of the Gro β or γ protein to anadditional sequence which does not destroy its activity or to additionalsequence cleaved under controlled conditions (such as treatment withpeptidase) to give an active protein, is obtained. If the sequence isuninterrupted by introns it is suitable for expression in any host. Ifthere are introns, expression is obtainable in mammalian or othereucaryotic systems capable of processing them. This sequence should bein excisable and recoverable form. The excised or recovered codingsequence is then placed in operable linkage with suitable controlsequences in a replicable expression vector. The vector is used totransform a suitable host and the transformed host cultured underfavorable conditions to effect the production of the recombinant Gro βor γ.

Genomic or cDNA fragments are obtained and used directly in appropriatehosts. The constructions for expression vectors operable in a variety ofhosts are made using appropriate replications and control sequences, asset forth below. Suitable restriction sites can, if not normallyavailable, be added to the ends of the coding sequence so as to providean excisable gene to insert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, or mammalian cells are presently usefulas hosts. Host systems which are capable of proper post-translationalprocessing are preferred. Accordingly, although procaryotic hosts are ingeneral the most efficient and convenient for the production ofrecombinant proteins, eucaryotic cells, and, in particular, mammaliancells are preferred for their processing capacity, for example, theability to form the proper glycosylation patterns. In addition, there ismore assurance that the native signal sequence will be recognized by themammalian host cell, thus making secretion possible, and purificationthereby easier.

2. Control Sequences and Corresponding Hosts:

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al., 1977, Gene, 2:95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences are defined herein to include promoters for transcriptioninitiation, optionally with an operator, along with ribosome bindingsite sequences, which include such commonly used promoters as thebeta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., 1977, Nature, 198:1056) and the tryptophan (trp)promoter system (Goeddel, et al., 1980, Nucleic Acids Res., 8:4057) andthe λ derived P_(L) promoter and N-gene ribosome binding site(Shimatake, et al., 1981, Nature, 292:128), which has been made usefulas a portable control cassette, as set forth in U.S. Pat. No. 4,711,845,issued Dec. 8, 1987. However, any available promoter system compatiblewith procaryotes can be used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used although a number of other strains are commonlyavailable. Examples of plasmid vectors suitable for yeast expression areshown in Broach, J. R., 1983, Meth. Enz., 101:307; Stinchcomb et al.,1979, Nature, 282:39; and Tschempe et al., 1980, Gene, 10:157 and aarke,L., et al., 1983, Meth.Enz., 101:300. Control sequences for yeastvectors include promoters for the synthesis of glycolytic enzymes (Hess,et al., 1968, J. Adv. Enzyme Reg., 7:149; Holland, et al., 1978,Biochemistry, 17:4900). Additional promoters known in the art includethe promoter for 3-phosphoglycerate kinase (Hitzeman, et al., 1980, J.Biol. Chem., 255:2073), and those for other glycolytic enzymes, such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters, which havethe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytocluome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and enzymes responsible for maltose and galactoseutilization (Holland, supra). It is also believed that terminatorsequences are desirable at the 3' end of the coding sequences. Suchteminators are found in the 3' untranslated region following the codingsequences in yeast-derived genes. Many of the vectors illustratedcontain control sequences derived from the enolase gene containingplasmid peno46 (Holland, M. J. et al., 1981, J. Biol. Chem., 256:1385)or the LEU2 gene obtained from YEp13 (Broach, J. et al., 1978, Gene,8:121), however, any vector containing a yeast compatible promoter,origin of replication and other control sequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, 1973, Cruz and Patterson, eds.,Academic Press. Useful host cell lines include murine myelomas N51, VEROand HeLa cells, and Chinese hamster ovary (CHO) cells. Expressionvectors for such cells ordinarily include promoters and controlsequences compatible with mammalian cells such as, for example, thecommonly used early and later promoters from Simian Virus 40 (SV40)(Fiers, et al., 1978, Nature, 273:113), or other viral promoters such asthose derived from polyoma, Adenovirus 2, bovine papilloma virus, oravian sarcoma viruses, or immunoglobulin promoters and heat shockpromoters. General aspects of mammalian cell host system transformationshave been described by Axel, U.S. Pat. No. 4,399,216, issued Aug. 16,1983. It now appears also that "enhancer" regions are important inoptimizing expression; these are, generally, sequences found upstream ofthe promoter region. Origins of replication may be obtained, if needed,from viral sources. However, integration into the chromosome is a commonmechanism for DNA replication in eucaryotes. Plant cells are also nowavailable as hosts, and control sequences compatible with plant cellssuch as the nopaline synthase promoter and polyadenylation signalsequences (Depicker, A. et al., 1982, J. Mol. Appl. Gen., 1:561) areavailable. Methods and vectors for transformation of plant cells havebeen disclosed in PC Publication No. WO 85/04899, published Nov. 7,1985.

Host strains useful in cloning and expression herein are as follows:

For cloning and sequencing, and for expression of construction undercontrol of most bacterial promoters, E. coli strain MM294 obtained fromE. coli Genetic Stock Center GCSC #6135. For expression under control ofthe P_(L) N_(RBS) promoter, E. coli strain K12 MC1000 lambda lysogen, N₇N₅₃ cI857 SusP80, a strain deposited with the Americana Type CultureCollection (ATCC 39531), may be used. E. coli DG116, which was depositedwith the ATCC (ATCC 53606) on April 7, 1987, may also be used.

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98, can be employed. The DG98strain has been deposited with the ATCC (ATCC 39768) on Jul. 13, 1984.

Mammalian expression can be accomplished in COS-A2 cells, COS-7, CV-1,hamster and murine cells. Insect cell-based expression can be inSpodoptera frugiperda.

3. Transformations:

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., 1972, PNAS (USA),69:2110, is used for procaryotes or other cells which containsubstantial cell wall barriers. Infection with Agrobacterium tumefaciens(Shaw, C. H. et al., 1983, Gene, 23:315) is used for certain plantcells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, 1987, Virology,52:546 is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P. et al., 1977, J. Bact.,130:946 and Hsiao, C. L. et al. 1979, PNAS (USA), 76:3829.

4. Probing mRNA by Northern Blot; Probe of cDNA or Genomic Libraries:

RNA is fractionated for Northern blot by agarose slab gelelectrophoresis under fully denaturing conditions using formaldehyde,Maniatas, T., et al. 1982, Molecular Cloning, Cold Spring Harbor Press,pp. 202-203, or 10 mM methyl mercury (CH₃ HgOH) (Bailey, J. M., et al,1976, Anal. Biochem., 70:75-85; Shegal, P. B. et al., 1980, Nature,288:95-97) as the denaturant. For methyl mercury gels, 1.5% gels areprepared by melting agarose in running buffer (100 mM boric acid, 6 mMsodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60°C. and adding 1/100 volume of 1 M CH₃ HgOH. The RNA is dissolved in0.5×running buffer and denatured by incubation in 10 mM methyl mercuryfor 10 minutes at room temperature. Glycerol (20%) and bromophenol blue(0.05%) are added for loading the samples. Samples are electrophoresedfor 500-600 volt-hr with recirculation of the buffer. Afterelectrophoresis, the gel is washed for 40 minutes in 10 mM2-mercaptoethanol to detoxify the methyl mercury, and Northern blotsprepared by transferring the RNA from the gel to a membrane filter.

cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Bacterial colonies, or the plaques for phage,are lifted onto duplicate nitrocellulose filter papers (S&S type BA-85).The plaques or colonies are lysed and DNA is fixed to the filter bysequential treatment for 5 minutes with 500 mM NaOH, 1.5 M NaCl. Thefilters are washed twice for 5 minutes each time with 5×standard salinecitrate (SSC) and are air dried and baked at 80° C. for 2 hours.

The gels for Northern blot or the duplicate filters for cDNA or genomicscreening are prehybridized at 25° to 42° C. for 6 to 8 hours with 10 mlper filter of DNA hybridization buffer without probe (0-50% formamide,5-6×SSC, pH 7.0, 5×Denhardt's solution (polyvinylpyrrolidine, plusFicoll and bovine serum albumin; 1×=0.02% of each), 20-50 mM sodiumphosphate buffer at pH 7.0, 0.2% sodium dodecyl sulfate (SDS), 20 μg/mlpoly U (when probing cDNA), and 50 μg/ml denatured salmon sperm DNA).The samples are then hybridized by incubation at the appropriatetemperature for about 24-36 hours using the hybridization buffercontaining kinased probe (for oligomers). Longer cDNA or genomicfragment probes were labelled by nick translation or by primerextension.

The conditions of both prehybridization and hybridization depend on thestringency desired, and vary, for example, with probe length. Typicalconditions for relatively long (e.g., more than 30-50 nucleotide) probesemploy a temperature of 42° to 55° C. and hybridization buffercontaining about 20%-50% formamide. For the lower stringencies neededfor oligomeric probes of about 15 nucleotides, lower temperatures ofabout 25°-42° C., and lower formamide concentrations (0%-20%) areemployed. For longer probes, the filters may be washed, for example,four times for 30 minutes, each time at 40°-55° C. with 2×SSC, 0.2% SDSand 50 mM sodium phosphate buffer at pH 7, then washed twice with0.2×SSC and 0.2% SDS, air dried, and are autoradiographed at -70° C. for2 to 3 days. Washing conditions are somewhat less harsh for shorterprobes.

5. Vector Construction:

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general about 1 μg ofplasmid or DNA sequence is cleaved by 1 unit of enzyme in about 20 μl ofbuffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about 1 hour to 2 hours at about 37° C.are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods of Enzymology, 1980,65:499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° to 25° C. in 50 mM dithiothreitol (DTT)and 5-10 μM dNTPs. The Klenow fragment fills in at 5' sticky ends butchews back protruding 3' single strands, even though the four dNTPs arepresent. If desired, selective repair can be performed by supplying onlyone of the, or selected, dNTPs within the limitations dictated by thenature of the sticky ends. After treatment with Klenow, the mixture isextracted with phenol/chloroform and ethanol precipitated. Treatmentunder appropriate conditions with S1 nuclease results in hydrolysis ofany single-stranded portion.

Synthetic oligonucleotides may be prepared by the triester method ofMatteucci et al., 1981, J. Am. Chem. Soc., 103:3185-3191, or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labelling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nmole substrate inthe presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM DTT, 1-2 mM ATP.If kinasing is for labelling of probe, the ATP will contain highspecific activity 32YP.

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml bovine serum albumin (BSA), 10 mM-50 mM NaCl, and either40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for "stickyend" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C. (for "blunt end" ligation). Intermolecular "sticky end" ligations areusually performed at 33-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). Intermolecular blunt end ligations (usuallyemploying a 10-30 fold molar excess of liners) are performed at 1 μMtotal ends concentration.

In the vector construction employing "vector fragments", the vectorfragment is commonly treated with bacterial alkaline phosphatase (BAP)in order to remove the 5' phosphate and prevent religation of thevector. BAP digestions are conducted at pH 8 in approximately 150 mMTris, in the presence of Na²⁺ and Mg²⁺ using about 1 unit of BAP per μgof vector at 60° C. for about 1 hour. In order to recover the nucleicacid fragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

6. Modification of DNA Sequences:

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis isused. This technique is now standard in the art, and is conducted usinga primer synthetic oligonucleotide complementary to a single strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells which harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form: 50% will have the original sequence.The plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

7. Verification of Construction:

Correct ligations for plasmid construction could be confirmed by firsttransforming E. coli strain MM294, or other suitable host, with theligation mixture. Successful transformants are selected by ampicillin,tetracycline or other antibiotic resistance or using other markersdepending on the mode of plasmid construction, as is understood in theart. Plasmids from the transformants are then prepared according to themethod of Clewell, D. B. et al., 1969, PNAS (USA), 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., 1972, J.Bacteriol, 110:667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al., 1977, PNAS (SA),74:5463 as further described by Messing et al., 1981, Nucleic AcidsRes., 9:309, or by the method of Maxam et al., 1980, Methods inEnzymology, 65:499.

E. General Methods of Purifying the Gro β or γ:

The general scheme for isolating and purifying the inflammatorycytokines consists of releasing the molecule from the cytoplasm of theappropriate cells, tissues or organs, followed by removing insolublematerial and subjecting the soluble fraction to one or morechromatographic steps including anion and cation exchangechromatography. Various purification methods may be used to purify theinflammatory cytokines. Regardless of the procedure chosen, anddepending on the nature of the biological material that the inflammatorycytokines are purified from, it may be desirable to have present in thevarious purification solutions one or more protease inhibitors, forexample, PMSF. Additionally, as is known in the art, certainpurification steps may be conducted at temperatures that reduce the riskof the inflammatory cytokines being proteolyzed.

More specifically, each inflammatory cytokine is prepared by releasingthe molecule from the cytosol using any number of techniques includingfreeze thawing, sonication, mild detergent extraction, etc. Thisprocedure is preferably carried out in a physiologically bufferedsolution containing one or more protease inhibitors. Moreover, tofurther inhibit protease activity, especially those proteases that relyon metal ions for activity, the extraction solution may contain metalion chelators. The preferred extraction solution is a physiologicallybalanced salt solution containing the chelatorsethyleneglycoltrichloroacetic acid (EGTA), or EDTA, plus the proteaseinhibitor phenylmethylsulfonylfluoride (PMSF). The metal ionchelator(s), as well as the protease inhibitor(s) are present atconcentrations that effectively inhibit proteolysis, preferably about 5mM and 100 μM, respectively. However, it will, of course, be appreciatedby those skilled in the art that since the types and amounts ofproteases vary depending on the starting material used to extract theinflammatory cytokine, the concentrations that the protease inhibitorsor chelators are used at, if indeed used at all, will also vary.

The mixture containing the inflammatory cytokine is clarified bycentrifugation, or in other ways to remove insoluble material from theaqueous cytosol fraction. If the cytosol fraction contains low amountsof the inflammatory cytokine, it can be concentrated by any one ofseveral techniques well known to those skilled in the art, includinghigh salt precipitation, such as, for example, with ammonium sulfate, orby ultra filtration. If the inflammatory cytokine is concentrated byprecipitation, it is preferably subsequently resuspended in a suitablephysiologically balanced salt solution containing protease inhibitor(s)and which can contain about 0.1% of a nonionic detergent. This solutionis then prepared for ion exchange chromatography by dialyzing it againsta compatibly buffered chromatographic solution, preferably containingmillimolar phosphate, a metal ion chelator, and a protease inhibitor.

The purification procedure would preferably employ at least one ionexchange chromatographic step. An example of a cation exchanger would beSP-cellulose cation exchanger. Such are commercially available from AMFMolecular Separations Division, Meridian, Conn. under the brand nameZetaPrep SP cartridges. The SP-cellulose cation exchanger is an elastic3-dimensional network composed of cellulosic backbones cross-linked withvinyl polymer containing pendant sulfopropyl functional groups. Thematrix is preferably adapted for radial flow passage of the inflammatorycytokine solution. The flow rate of the solution through the matrix willdepend upon the size and geometry of the matrix used. It will beapparent to those skilled in the art, however, that care should be takento avoid exceeding the unit capacity of the matrix with the inflammatorycytokine. If the capacity is exceeded, the inflammatory cytokine willnot be totally retained and excess unretained inhibitor will be presentin the effluent. Examples of anion exchange chromatographic materialswhich can be used are DEAE-Sepharose (Pharmacia Corp.) andTSK-DEAE-5-PW.

As mentioned above, the initial sequence of chromatographic proceduresis not rigid, and can be varied. In addition to anion exchangechromatography, other chromatographic methods may be employed, alone orin combination, which are known and practiced in the art. Exemplarymethods would include affinity chromatography using, preferably,antibodies specific for the inflammatory cytokines.

The methods for eluting proteins from anion exchangers, and affinitycolumn are well documented and generally known in the art. For example,the inflammatory cytokines can be eluted from DEAE suing a suitablybuffered salt gradient, while in the latter instance the addition of achaotropic agent may be effective. The salt gradient and theconcentration of chaotrope can be determined empirically.

A third chromatographic technique that may be used alone or incombination to purify the inflammatory cytokines is by hydrophobicinteraction chromatography. A variety of hydrophobic interactionchromatographic matrixes may be utilized. Generally, the materials andmethods for utilizing hydrophobic chromatography are described by S.Shaltie, 1984, Methods in Enzymology, 104:69. The preparation of suchsolutions is within the skill of the art.

The purity of the inflammatory cytokines so obtained can be monitored bysodium dodecyl sulfate polyacrylamide gel electrophoresis run underreducing conditions. The general procedures for preparing and using theabove purification materials are known to those skilled in the art.

F. General Uses of Gro β and Gro γ:

As shown in Example 1 below, the Gro β and γ genes were expressed in thepresence of inflammatory agents. Without wishing to be bound by thefollowing postulation, it is postulated that treatments utilizing Gro βand γ would prevent or alleviate the severity of inflammation and thedisease or conditions associated therewith.

Example 1 also show that Gro γ was found in colonic epithelial tumorcells but not in adjacent normal epithelial cells. As a comparison, ithas been observed that Gro α is over-expressed in other tumor cell linessuch as CHEF/16 cells, src-transformed chicken fibroblasts, and humanmelanomas. On the other hand, it has also been observed that Gro α isexpressed in normal growing mammary cells but was absent in manycarcinomas. (Anisowicz, A et. al., 1988, PNAS (USA), supra.) It appearsthat the differential expression of Gro βor γ in different cell linesmay affect the transformation of these cells. Therefore, depending onthe cell and tumor types in question, the tumor treatment regimen wouldinvolve varying the amount of Gro β or γ accessible to the cells. Thiscan be achieved by either increasing or decreasing the amounts of theGro proteins available to the cells, depending on whether thetumorigenesis is due to their over- or under-expression of the Grogenes.

Having described what the applicants believe their invention to be, thefollowing examples are presented to illustrate the invention, and arenot to be construed as limiting the scope of the invention. For example,variation in: the source of the inflammatory cytokines; the method forinducing, activating and in general producing and obtaining theinflammatory cytokines and antibodies against them; diagnostic testsusing them; detection tests for them may be employed without departingfrom the scope of the present invention.

EXAMPLE 1

A. Procedure

1. Construction and Screening of the Adherent Monocyte cDNA Library forAnalogues of Gro:

The adherent monocyte cDNA library was prepared in the bacterialexpression vector λgt10 by using total RNA from human blood monocytesadhered to plastic for 30 minutes at 37° C. The cDNA library used inthis application, the construction and screening of the library wasperformed as described in Sporn, S. A., J. of Immunol., supra. Theprocedure was as follows.

a) Isolation of Monocytes

Monocytes were isolated from platelet-phoresis residue bags obtainedfrom the American Red Cross (Durham, N.C.). Mononuclear cells were firstisolated by Ficoll-Hypaque sedimentation according to the proceduredescribed in Boyum, A., 1986, Scand. J. Clin. Lab. Invest., 21:77 andwashed three time with Versene (GIBCO, Grand Island, N.Y.) to removeplatelets. Monocytes were then isolated by Percoll density fractionationas described in Ulmer, A. J., et al., 1979, J. Immunol. Methods, 30:1 orby adherence to plastic tissue culture dishes (Corning 25020, Corning,N.Y.) as described in Eierman, D. F., et ., 1989, supra.

b) Cell Culture

Monocytes were cultured in endotoxin-free RPMI 1640 (Cell-gro, FisherScientific, Raleigh, N.C.) either adherently on plastic at 1 to 2×10⁷cells/dish or nonadherendy in 50 ml polypropylene tubes (Fisher) at 10⁶cells/ml (30 ml/tube). Monocytes were cultured serum-free, unlessotherwise indicated, at 37° C. in 5% CO₂. The coating of plates withfibronectin (FN), collagen, or FN/anti-FN complexes was performed asdescribed in Eierman, D. F., et al., 1989, supra.

c) Construction of a cDNA Library

Total RNA isolated from monocytes adhered to plastic for 30 minutes wasused for construction of a cDNA library by using modified methods ofWatson and Jackson (Watson, C. J, et al., 1985, in DNA Cloning, Vol. 1,A Practical Approach, Glover, D. M. ed., IRL Press, Oxford, England, p.79) and Huynh, T. V. et al., 1985, in DNA Cloning, Vol. 1, A PracticalApproach, Glover, D. M., ed., IRL Press, Oxford, England, p. 49). RNAwas converted to dscDNA by using avian myeloblastosis virus reversetranscriptase and the klenow fragment of DNA polymerase I. dscDNAfragments with EcoR I linkers were size-selected and packaged into λgt10 with the use of Gigapack (Stratagene, San Diego, Calif.). Theunamplified library contained approximately 5.3×10⁶ recombinants at afrequency of 7×10⁷ /μg DNA.

Fifty thousand plaques from the original library were grown and plaquelifts were hybridized to a ³² P-labelled first strand cDNA probes madefrom time 0 (uninduced) monocyte RNA. In order to reduce the number ofclones in the screening that are expressed in uninduced monocytes, 4,000clones that did not hybridize to the time 0 probe were selected andpooled to form a sub-library. The original library and subsequently thesub-library were hybridized with an IL-1β probe, which is an example ofa gene induced by adherence. This demonstrated a 3- to 4-fold enrichmentfor the induced clones in the sub-library and a depletion of greaterthan 80% of clones common to adhered and nonadhered monocytes.

d) Screening of the cDNA Library

The sub-library was screened by differential hybridization with ³²P-labelled total first-strand cDNA probes using the procedure describedin Huynh, T. V., et al., 1985, supra, prepared from RNA from either30-minute and 4-hour adhered or nonadhered monocytes. Plaques thatpreferentially hybridized with the total cDNA probes made from adheredmonocytes as opposed to non-adherent monocytes were selected andrescreened. A total of 35 clones out of 111 original isolates remainedpositive after the second screening.

e) Reagents

PMA, E. coli serotype 055:B5 LPS, the calcium ionophore A23187, andhuman collagen type I were obtained from Sigma Chemical Co. (St. Louis,Mo.). Human FN and anti-human FN were obtained from CollaborativeResearch (Bedford, Mass.).

f) Isolation of RNA and Northern Analysis

Total RNA was isolated by using the guanidine isothiocyanate-cesiumchloride method as described in Chirgwin, J. M., et al., 1979,Biochemisty, 18:5294. Total RNA (15 μg/lane) was denatured and subjectedto electrophoresis on 1% agarose gels containing formaldehyde. RNA wassubsequently blotted by Northern transfer (as described in Thomas, P.S., 1980, PNAS USA), 77:5201) onto nitrocellulose. Clone cDNA insertswere excised from λgt10 vector, ³² P-labelled by nick translation andused as probes for hybridization of Northern blots. The hybridizationswere performed at 42° C. as described in Haskill, et al., 1988, supraand blots were washed to a stringency of 0.2×SSC at 56° C.

g) DNA Sequencing

cDNA inserts were subcloned into the double stranded vector pGEM blue(Promega Biotec, Madison, Wis.). dsdDNA sequencing was performed by thedideoxy chain termination method (as described in Sanger, F. S., et al.,1977, PNAS (USA), 74:5463) by using the Sequenase sequencing kit (UnitedStates Biochemical Co., Cleveland, Ohio) with T7 and SP6 primers(Promega), as well as sequence-specific oligonucleotide primers (CetusCorp., Emeryville, Calif.).

h) Southern Blot Hybridization

Human monocyte DNA was isolated (as described in Maniatis, T., et al.,1982, Molecular Cloning, Cold Spring Harbor Lab., Cold Spring Harbor,N.Y., p. 382) and digested with the restriction enzymes indicated. Thedigested DNA was electrophoresed on a 1% agarose gel, denatured, andtransferred to nitrocellulose (as described in Southern, E. M., 1975, J.Mol. Biol., 98:503).

B. Result

A 880 bp partial clone (C2m) (designated MAD-2 in Sporn et al., 1990,supra) isolated by subtractive hybridization showed some sequencesimilarity to the original Gro but contained a number of changes in the3' coding region. This clone was used to isolate 5 additional clonesfrom a second library produced from mezerin and calcium ionophorestimulated leukocytes, the procedure is described below.

A. Procedure

1. cDNA Library Produced From Mezerein and Calcium Ionophore StimulatedLeukocytes

The cDNA was generated from RNA obtained from induced peripheral bloodlymphocytes. Such procedures are well known in the at The preferredprocedure consists of inducing peripheral blood cells for three dayswith a calcium ionophore and mezerein. The preferred ionophore isA-23187. The induction procedure is generally described in U.S. Pat. No.4,376,821, issued Mar. 15, 1983, "Production of Human IFN-Gamma (Immune)Interferon," to I. A. Braude and U.S. Ser. No. 517,276, filed May 1,1990 by Haskill et al., "Interleukin-1 Antagonist and Uses Thereof".

More specifically, leukocytes were induced with 100 ng/ml of mezereinand 0.25 μg/ml of calcium ionophore A-23187. The induction period wasabout 3 days, after which the total RNA was isolated from the inducedperipheral blood lymphocytes using essentially the guanidineisothiocyanate-cesium chloride described in Chirgwin et al., 1979,supra, and the poly (A+) messenger RNA were isolated from the total RNAfraction by chromatography on oligo (dT) cellulose as described in Avivet al., 1972, supra.

Next, the first strand of the cDNA was obtained by reverse transcribingthe mRNA as follows. 33 μg of the poly (A+) RNA was dispensed into amicro-centrifuge tube, and incubated for 5 minutes at 65° C., and thencooled on ice for 5 minutes. Next, to the RNA was added sequentially thefollowing: 66 μl of 250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM of MgCl₂; 10.0 mM DTT; 9.0 μl RNA's in [40 U/μl]; 16.5 μl of 10 mM dNTP's (i.e.,10 mM dATP+10 mM dCTP+10 mM dGTP+10 mM dTTP); 35.0 μl of p(dT)₁₂₋₁₈ (100pmole/μl) (p(dT)₁₂₋₁₈ denotes oligodeoxythymidilic acid population withlengths of 12 mer, 13 mer, etc., up to 18 mer); and 16.5 μl of murineleukemia virus (MLV)-reverse transcriptase (200 U/μl), and water tobring to a total volume of 330 μl.

To monitor the efficiency of first strand synthesis by alkaline agarosegel electrophoresis, a second tube was prepared that contained 2 μl [α³²P] dCIP (10 μCi/μl), and 20 μl of the first strand reaction mixtureprepared as described above.

Both tubes were incubated for 60 minutes at 37° C. followed by stoppingthe reactions by putting the tubes on ice. Five μl of 0.5 M EDTA and 23μl of water was added to the tube containing [α³² P] dCTP. Both tubeswere stored at -20° C.

The second strand of the cDNA duplex was prepared as follows. 37.5 μl ofthe first strand mixture synthesized as described above was aliquotedinto 8 separate tubes, on ice. Also, 6.25 μl of the mix was pipettedinto a 9th tube, on ice. Next, to each of the 8 tubes, 262.5 μl of asecond strand cocktail was added, and to the remaining, or control tube,43.75 μl was added. The second strand cocktail consisted of thefollowing: 600.0 μl 5× second strand buffer (SSB) (5×SSB consisted of 94mM Tris-HCl pH 8.3,453 mM KCl, 23.3 mM MgC₂, 18.7 mM DTT) 1802.9 μlwater, 56.3 μl 10 mM dNTP's, 12.5 μl [α³² P] dCTP, 75.0 μl 6 mM β-NAD,75.0 μl DNA polymerase I (10 u/μl), and 3.4 μl of E. coli ligase (9u/μl). The total volume of the reagents in the second strand cocktailwas 2,625.0 Al. All 9 tubes were incubated for 2 hours at 16° C., afterwhich to the 9th tube was added 5 μl of 0.5 M EDTA and 65 μl of water.The contents of this tube were stored at -20° C. for subsequent alkalineagarose gel analysis.

Next, the contents of the 8 tubes were phenol extracted and twiceethanol precipitated, and the pellets combined in 128 μl of TE.

The cDNA prepared above was treated with Rnase H as follows. To 128 μlof cDNA was added the following: 128 μl 5×Rnase H buffer which consistedof 100 mM Tris-HCL, pH 7.5, 100 mM KCl, 50 mM MgCl₂, 0.7 mM DTT, and 0.5mM EDTA. Additionally, the tube contained 377.3 μl water, and 6.7 μl ofRnase H (1.9 u/μl). The total volume of the Rnase H reaction digest is640 μl. The mixture was incubated at 37° C. for 20 minutes after which 5μl of 0.5 M EDTA was added to terminate the reaction. Finally, thereaction mixture was phenol extracted, twice ethanol precipitated, andthe pellet resuspended in 12 μl of TE.

The cDNA was fractionated and purified by neutral agarose gelelectrophoresis. cDNAs of about 0.25-7.0 kilo bases in length wereremoved from the gels, glass bead purified, and resuspended in 107.5 μlof glass distilled water.

Next, the cDNA was C-tailed, and cloned into the vector pCDLSRα-296[obtained from DNAX corporation, and described by Takebe et al., 1988,Molecular and Cellular Biology, 8(1):466; and in U.S. Pat. No.4,695,542] as follows. The 10×terminal deoxynucleotidyl transferasebuffer (hereinafter referred to as 10×TdT buffer) was prepared asfollows: 13.8 g cacodylic acid was added to 3.0 g Tris-base in 60 ml ofwater. The solution was adjusted to pH 7.6 by slow addition of solidKOH, after which the volume was increased to 88 ml with water.Subsequently, the solution was chilled to 0° C., then 2 ml of 0.1 M DTTwas added followed by the addition of 10 ml of 0.1 M MnCl₂ dropwisewhile the solution was being constantly stirred. To the 38.3 μl of10×TdT buffer was added 5.7 μl of 1 mM dCTP, and 0.6 μg of doublestranded cDNA. An amount of water was added to bring the solution to atotal volume of 380.5 μl. The solution was warmed to 37° C. for 15minutes, and 360 units of TdT in about 3 μl was added. 60 μl aliquot wasremoved at various times and combined with 468 μl of 1 mM EDTA. Eachaliquot was then combined with previously G-tailed vector in a suitableamount of annealing buffer, and transformed into an appropriate host.The C-tailed stock that produced the greatest number of transformantswas used for the large scale transformation.

The cDNAs were cloned into the plasmid vector, pCDL-SRα296. The vectorwas prepared as follows. 200 μg of the plasmid was digested with 700units PstI in 500 μl total volume, for 60 minutes at 37° C. The plasmidwas then phenol extracted, and twice ethanol precipitated, andresuspended in 200 μl of distilled water. The concentration wasdetermined by spectrophotometry. Next, about 136 μg of pCDL-SRα296,112.6 pmole 3' ends, was G-tailed by combining it in a solutionconsisting of 30 μl of 10×TdT buffer, 30 μl [³ H] dGTP (approximately 70pmole/μl), 5.5 μl of 1 mM dGTP and water to make a total volume of 297μl. The tailing reaction was conducted for various times, and theprocedure consisted of removing 30 μl from the reaction tube andcombining it with 359 μl of 17 mM EDTA, which was the 0 time point.Next, 360 units of terminal transferase in 3 μl was added to prewarmedreaction digest mixture, 15 minutes at 37° C., and 30 μl of the mixtureremoved at various time intervals and the reactions stopped by pipettingit into 359 μl of 17 mM EDTA. The amount of G-tailing was monitored bydetermining the incorporation of [³ H] dGTP, as is known in the art. Toensure that the pCDL-SRα296 vector was properly G-tailed and that it wasnot contaminated with untailed vector, a trial annealing andtransformation of DH5α bacteria was conducted as is known in the art.(DH5α bacteria was obtained from Bethesda Research Laboratories,Research Products Division, Life Technologies, Inc., Gaithersburg, Md.20877. Cat. No. 82585A: MAX EFFICIENCY DH5α™ competent cells.) Thevector was stored at -20° C. and used to clone the above describedC-tailed cDNA.

Briefly, cloning into pCDL-SRα296 consisted of combining 88 μl ofC-tailed cDNA corresponding to each time point, 2 μl G-tailed vector,and 10 μl of 10×annealing buffer. The total volume was 100 μl. The10×annealing buffer consisted of: 0.1 M Tris-HCL, pH 7.6, 1.0 M NaCl, 10mM EDTA. The annealing reaction was conducted under standard conditions,and the mixture transformed into DH5 bacteria.

The cDNA library obtained above may be amplified using either procedureswell known in the art, or a novel solid state amplification techniquedescribed as follows. The procedure consisted of suspending bacterialtransformants in low melting temperature agarose. This is contrary tothe state of the art methods that plate the bacterial transformants onsuitable culture dishes. The following materials and methods wereutilized 0.3% Seaprep agarose in 2×LB media (2% bactotryptone, 1% yeastextract 1% NaCl, pH 6.9. The bactotryptone and yeast extract wereobtained from Difco Laboratories, Inc., Detroit, Mich.), maintained at37° C. in a water bath. An appropriate amount of cDNA annealed topCDL-SRα296 and transformed into DH5α bacteria to generate up to about2.5×10⁶ cfu/ml. To the appropriate amount of agarose was addedampicillin to make 50 μg/ml and approximately 1.25×10⁶ cfu of bacterialtransformants. 25 ml of this solution was poured into 50 ml conicaltubes (Falcon Corp. No. 2098). The tubes were placed in an ice waterbath for 20-60 minutes, and incubated overnight at 37° C. To assess thetitre of the transformant mixture, 100 μl was plated on LB-amp⁵⁰ (amp⁵⁰denotes 50 μg/ml ampicillin) plates.

The library may be stored by pelleting the cells in 500 ml centrifugebottles at 8 K for 20 minutes at room temperature. The cell pellets wereresuspended in a total volume of 100 ml of 12.5% glycerol in LB mediaAliquots of the suspension were stored at -70° C.

2. Origin and Culture of Cells:

Monocytes and lymphocytes were isolated from normal blood byFicoll-Hypaque and subsequent Percoll density separation as describedpreviously (Eierman, D. F., et al., 1989, supra . Neutrophils werepurified by sedimentation of the Ficoll-Hypaque pellet in a 3%gelatin/PBS (phosphate buffered saline solution without Ca++ and Mg++)for 1 hour at 37° C. Monocytes, lymphocytes and neutrophils werecultured in serum-free RPMI medium (GIBCO, Grand Island, N.Y.) onplastic dishes which had previously been coated with fibronectin, or onplastic with 1 μg/ml bacterial endotoxin lipopoly-saccharide (LPS) orwere cultured under non-adherent conditions with 5 ng/ml of a knowntumor promoter phorbol 12-myristate 13-acetate (PMA) (Eierman, D. F., etal., 1989, supra). Second passage human umbilical vein endothelial cells(HUVE) were cultured in 10% fetal calf serum in RPMI medium for 4 hourswith 5 μg/ml LPS. Colon carcinoma cells were derived from a freshsurgical specimen. Diagnosis was confirmed by histology.

3. Isolation of Genomic Clones:

Approximately 8×10⁵ plaques of a human female leukocyte library in λEMBL 3 were screened with ³² P labelled Gro α cDNA probe. Positiveclones were subcloned into pGEM3 or pGEM4Z plasmids and the wholeplasmid or ExoIII nested deletion plasmids were sequenced by the dideoxychain termination method. The three Gro gene clones were distinguishedby restriction fragment analysis.

4. Analysis of RNA and DNA:

RNA analysis was carried out by PCR. For PCR, 1 μg of total RNA wasconverted into first strand cDNA with random hexamers as described byKawasaki, E. S., et al., 1990, PCR Technology, (Erlich, H. A., ed.)Stockton Press, New York, pp. 89-97. Amplification was carried out usinga sense primer that was common to the different Gro clones andanti-sense primers specific for each. Gro α was detected with GM135 andGM350, Gro β with ML80 and GM272; and Gro γ with GM135 and GM221 (seeFIGS. 1(a) and (b)).

Amplifications were carried out to 25, 30 and 35 cycles to verify thatreactions were proceeding exponentially. Data shown is from 30 cycles atwhich time all samples had not yet reached maximum intensity. Southerntransfer analysis was carried out following transfer to nitrocellulosefor random primed cDNA probes or by direct oligonucleotide hybridizationto dehydrated agarose gels as described in Southern, E. M, 1975, supraand Siebert, P. D., et al., 1989, Clontech Laboratories 1989 TechnicalReport. 6-9. Washing was carried out to a final stringency of 2×SSPE(2×SSPE consisted of 0.3 M NaCl, 0.02 M NaH₂ PO₄, and 0.002 M EDTA) at56° C. for oligonucleotides and 0.2×SSC, at 65° C. for cDNA probes.

B. Results

1. Characterization of Two Novel Gro Gene cDNAs:

During isolation of cDNAs by substrative hybridization from adherent vs.non-adherent monocytes (described in Sporn, S. A., et al., 1990, supra),a partial clone with a Gro-related sequence was identified, and usingthis sequence as probe against the leukocyte cDNA library above, fulllength versions of two sequences, designated Gro β and Gro γ wererecovered.

In summary, one of the 5 clones, isolated from the above mezerin andcalcium ionophore stimulated leukocyte cDNA library, was designated Groβ. Gro β contained a PstI and NcoI restriction pattern similar to theC2m isolate. Primers GM135, GM221, GM297, GM298, GM316, GM349, GM350 andML80 (shown in FIGS. 1(a) and (b)) were constructed which permittedbidirectional sequencing of the double strand templates. In the case ofGro β, a partial clone (C2m) (Designated MAD-2 in Sporn, S. A., et al.,1990, J. of Immunol., supra), a full-length clone containing 2 introns(CC2b) and polymerase chain reaction (PCR) product were used to confirmthe complete cDNA sequence.

A partial description of the Gro β gene is inferred from sequencing ofCC2b. The result showed close nucleic acid homology between CC2b and Groα and Gro γ at the 5' end up to the start of the mature protein.Thereafter, 95 nucleic acids intervened before the homology began againover the first 41 amino acids of the mature protein. Then anotherinsertion of 118 nucleic acids intervened before the homology beganagain for another 84 amino acids. Without wishing to be bound by thefollowing postulation, we postulate that the 95 and 118 nucleic acidfragments without homology to Gro α and Gro γ cDNAs represent introns ofthe Gro β gene, and that this particular cDNA, CC2b, represents afailure of the cell to completely process out the introns.

Another clone (Gro γ) was represented 3 times in this screening. Theremaining isolate appeared to have PstI and NcoI restrictioncharacteristics of the original Gro clone Gro α. All three different(i.e., Gro α, β and γ) clones were sequenced and their cDNA sequencesare shown in FIGS. 1(A) and (B).

Sequence comparisons of the α, β and γ forms are also given in FIGS.1(A) and (B). Gro β and Gro γ share 93% and 82% identity, respectively,with Gro α at the nucleotide level. Compared with Gro α, there are 11amino acid substitutions in Gro β, 8 resulting from first and/or secondposition changes. Of the 11, 9 are located in the secreted peptidebeyond the signal peptide cleavage point.

Compared with Gro α sequence, there are 15 amino acid changes in Gro γ,11 of which are in the secreted peptide region. In addition, there is acodon deletion in Gro γ at bp157.

In the 3' untranslated region, Gro β has 11 ATTTA repeats, 6 of them ina single stretch at bp543. In contrast, Gro α and Gro γ contain 5 and 6repeats, respectively. In addition, a 64 bp region and a 58 bp region 3'to it are highly conserved among the 3 Gro variants (beginning at bp847,870 and 852, in Gro α, β and γ respectively). The intervening 25 bp areconserved in Gro α and β. The 3'58 bp of the region (930-987) are alsoconserved in the hamster gene, as previously shown (Anisowicz, A., etal., 1987, supra).

2. DNA Hybridization Analysis Identifies Three Gro Genes:

Southern blot hybridization with Gro α, β, and γ cDNA probes gavesimilar restriction fragment patterns with the enzymes XbaI and EcoRI,as well as extra bands charactistic of each cDNA. In FIG. 2, thesepatterns (lanes 1, 2 and 3) were compared with Southern blots usingoligonucleotide probes common to the three cDNAs (lanes 4 and 5) as wellas Gro α-specific (lane 6) and β-specific (lane 7) probes. In the EcoRIdigests, the common probe ML80 identified three bands at 6.6, 4.4 and3.3 kb. (The sequence for ML80 is as shown in FIG. 1(B).) With the Groα-specific probe GM350, only the 4.4 kb band was seen (lane 6); and withthe β-specific probe GM272 only the 3.3 kb band was seen. ML80 alsodetected the 6.6 kb γ-specific DNA seen only in lane 3. Similarly in theXbaI digests the results are consistent in identifying three genes.

3. Expression of the Three Gro Genes is Differentially Regulated in aTissue and Signal Specific Manner:

Utilizing PCR primer pairs specific for each of the 3 Gro cDNAs, theexpression of these genes was characterized in (1) inflammatory cellsfrom a single donor (neutrophils, lymphocytes and monocytes); (2)umbilical vein endothelial cells; and (3) a freshly isolated coloncarcinoma from a different donor (FIG. 3). Neutrophils which adhered tofibronectin produced only the Gro α version. Lymphocytes produced Gro αonly, whereas monocytes expressed all three Gro molecules. Thus,different cells from the same donor stimulated by adherence to the sameextracellular matrix component showed selective Gro gene expression.

However, in the same cell type, different inducers stimulated differentpatterns of Gro expression. Non-adhered monocytes stimulated with PMAexpressed only the Gro β and Gro γ forms while monocytes treated withLPS expressed all 3 versions. Human umbilical vein endothelial cellsstimulated with LPS expressed only the Gro α gene product. Expression ofthe Gro γ gene was not restricted to macrophages; freshly isolated anddissected colonic epithelial tumor cells were found to expresspredominantly the Gro γ version by PCR analysis. In the same study, thenormal colonic epithelial cells adjacent to the tumor cells did notexpress detectable level of Gro γ.

These results confirm that all three forms of Gro α are expressed in theinduced cells. However, the results do not provide a precisequantitative comparison, since the relative hybridization constants forthe three probes may differ.

EXAMPLE 2 Methods for Identifying or Detecting Inflammatory andAnti-inflammatory Agents

A. Procedure

1. Methods for Identifying or Detecting Inflammatory Agents:

As Example 1 indicates, the monocytes under the following conditionsexpressed Gro β or γ. 1) monocytes which adhered to fibronectin; 2)non-adhered monocytes stimulated with PMA; and 3) non-adhered monocytestreated with LPS. Since PMA stimulates Gro β and γ expression innon-adhered monocytes, this indicates that the expression of these Grogenes were due to the presence of inflammatory agents, such as PMA andLPS.

Thus, an assay system may be established for screening potentialchemicals that may modulate the synthesis, release, or activity of theinflammatory cytokines. More particularly, the assay could be used todetermine if a chemical X could be an inflammatory agent. By varying theconcentration of X and assaying the levels of expression of Gro β and γ,it could also be determined at what concentration X would trigger aninflammatory response, and the potential severity of the inflammatoryresponse at different concentrations. For example, a sample of monocytescould be exposed to X in various amounts, preferably under nonadherentconditions, for an appropriate period of time to allow for theexpression of the Gro β or γ gene. Then the RNAs from the monocytes arecollected and probed with DNA probes for Gro β and γ, using thehybridization or PCR procedures described under the Procedure section ofExample 1 (A)(4). In situ hybridization could also be used using methodsknown in the art. The procedures for obtaining, culturing and incubatingthe monocytes with X would be similar to those set forth under theProcedure section of Example 1, for monocytes exposed to LPS on PMN. Oneskilled in the art would be able to appropriately modify the proceduresand the incubation time for the particular X used. This test would beuseful if X has been proposed as a potential drug, as it would alert thepotential users of its inflammatory effect and the prop precautions tobe taken.

Alternatively, the Gro β and γ could be detected using the immunologicalprocedures generally described supra.

2. Methods for Identifying or Detecting Anti-Inflammatory Agents:

The methods shown above could be modified to identify or detectanti-inflammatory agents. Thus, an assay system may be established forscreening potential chemicals that may modulate the synthesis, release,or activity of the inflammmatory cytokines. More particularly, the assaycould be used to determine if a chemical Y could be an anti-inflammatoryagent. The procedure would involve exposing a sample of monocytes to aknown inflammatory agent, such as PMS or LPS, for a sufficient time forthe expression of Gro β or γ. Then Y is added to the culture. After anappropriate period of incubation, the RNAs from the monocytes arecollected and purified and probed with DNA probes for Gro β or γ, usingthe procedure described under the Procedure section of Example 1(A)(4).In situ hybridization could also be used. Alternatively, the Gro β and γprotein could be detected using the immunological procedures generallydescribed supra. By varying the concentration of Y and assaying thelevels of expression of Gro β and γ, it could also be determined at whatconcentration Y is effective as an anti-inflammatory agent.

The procedures for obtaining, culturing and incubating the monocyteswith X would be similar to those set forth under the "Procedure" sectionof Example 1, for monocytes exposed to LPS on PMN. One skilled in theart would be able to appropriately modify the procedures for theparticular Y used and the appropriate incubation times for theinflammatory and potential anti-inflammatory agents. The methods areuseful in identifying anti-inflammatory agents and finding novelanti-inflammatory agents. The methods can also be used to determine theefficacy of and the appropriate dose levels for novel or knownanti-inflammatory agents.

EXAMPLE 3 Diagnostic for Colonic Epithelial Tumor Cells

As shown in Example 1, Gro γ was found in colonic epithelial tumor cellsbut not in adjacent normal epithelial cells. Therefore, colonicepithelial tumor could be diagnosed by assaying for the differentialexpression of Gro γ gene in the colonic epithelial cells. A preferredassay procedure would involve the hybridization of mRNA, collected fromthe epithelial cells to be tested, to a DNA probe for Gro γ. In situhybridization could also be used. The preferred procedure is thehybridization or PCR procedure described under the Procedure section ofExample 1 (A)(4). The preferred DNA probe would be GM221.

EXAMPLE 4 Synthesis of Peptide Sequences

The peptides containing the sequences of Gro β and γ may be synthesizedby methods well known in the art. The preferred method of peptidesynthesis is the solid-phase method, described in more detail inMerrifield, R. B., 1985, Science, 232:341-347, on a Biosearch 9500automated peptide machine, cleaved with hydrogen fluoride and purifiedby preparative HPLC using a Waters Delta Prep 3000 instrument, on a15-20 μm Vydac CH Prep PAK column. An alternative method is by means ofABI Automatic Synthesis. These synthetic peptides can be used to raisethe polyclonal and monoclonal antibodies to Gro β and γ (see Example 5below).

EXAMPLE 5 Production of Antibodies to Gro β and γ

Antibodies to the inflammatory cytokines are produced using standardprocedures known in the art. For example, antibodies are produced byinjecting a host animal such as rabbit, rat, goat, mouse, etc., with theGro β and γ proteins, or peptide fragments thereof, alone or conjugatedto an appropriate carrier if required to elicit an antibody response.Examples of the preferred peptides for raising antibodies against theseGro proteins are:

Cys-(Gly)₁₋₃ -5'-Asn;

Cys-(Gly)₁₋₃ -5'-Gly; and

Cys-(Gly)₁₋₃ -5'-(Gly)₁₋₃ -5'-Gly.

For Gro β, the 5' mer would be: Lys-Asn-Gly-Lys-Ser. For Gro γ, the 5'mer would be: Lys-Lys-Gly-Ser-Thr. The term (Gly)₁₋₃ denotes one tothree glycine residues.

Further, Gro β and γ, or peptide fragments can be combined with anadjuvant, for example, complete Freund's adjuvant, and used to immunizethe host animals. Alternatively, before injection into the host animals,these proteins or peptides may be conjugated with keyhole limpethemocyanin (KLH) or bovine serum albumin (BSA). The conjugation isachieved via the sulfhydryl group in the cysteine residue. The methodsfor such conjugation and the production of antibodies using theconjugate thus produced are described in U.S. Pat. No. 4,762,706, issuedAug. 9, 1988, to McCormick et al. It will be appreciated by thoseskilled in the art that monoclonal antibodies (MABs) to the above Gro βand γ are produced by means of the hybridoma technique. These monoclonalantibodies are useful for assaying the Gro proteins in an immunoassay asshown above.

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

We claim:
 1. A method for purifying Groβ, comprising the steps of(A)binding an anti-Groβ antibody to a matrix; (B) contacting a solutioncontaining Groβ with the matrix to allow binding of the Groβ to theanti-Groβ antibody to form an antigen-antibody complex; and (c)collecting Groβ from the antigen-antibody complex; wherein the Groβconsists of the amino acid sequence shown in lines designated "β" ofFIG. 1(B); wherein the anti-Grop antibody binds to a peptide selectedfrom the group consisting of(i) Cys-(Gly)₁₋₃ -Lys-Asn-Gly-Lys-Ser-Asn;(ii) Cys-(Gly)₁₋₃ -Lys-Asn-Gly-Lys-Ser-Gly; and (iii) Cys-(Gly)₁₋₃-Lys-Asn-Gly-Lys-Ser-(Gly)₁₋₃ -Lys-Asn-Gly-Lys-Ser-Gly; and wherein(Gly)₁₋₃ denotes one to three glycine residues.
 2. A method forpurifying Groγ, comprising the steps of(a) binding an anti-Groγ antibodyto a matrix; (b) contacting a solution containing Groγ with the matrixto allow binding of the Groγ to the anti-Groγ antibody to form anantigen-antibody complex; and (c) collecting Groγ from theantigen-antibody complex; wherein the Groγ consists of the amino acidsequence shown in lines designated "γ" of FIG. 1(B); wherein theanti-Groγ antibody binds to a peptide selected from the group consistingof(i) Cys-(Gly)₁₋₃ -Lys-Lys-Gly-Ser-Thr-Asn; (ii) Cys-(Gly)₁₋₃-Lys-Lys-Gly-Ser-Thr-Gly; and (iii) Cys-(Gly)₁₋₃-Lys-Lys-Gly-Ser-Thr-(Gly), ₃ -Lys-Lys-Gly-Ser-Thr-Gly; and wherein(Gly)₁₋₃ denotes one to three glycine residues.